PROJECT SUMMARY Somatic cell nuclear transfer (SCNT) allows for the creation of genetically identical animals for agricultural purposes, and is a powerful tool for production of genetically engineered animals for biomedical applications and generation of patient-specific embryonic stem cells (SCNT-ESCs) for therapeutics. However, the low efficiency of full term development limits its wide-spread use and practical utility. The fundamental problem is how the egg incompletely reprograms a somatic donor nucleus. The overall objectives of the current proposal are outlined in the two Specific Aims : 1) Identify epigenetic, chromatin, and transcriptomic landscapes in in vitro fertilized (IVF) embryos and then elucidate how SCNT embryos deviate from this developmental paradigm; and 2) Establish and test new approaches to correct reprogramming defects and improve SCNT development both in preimplantation and postimplantation embryos. We have created high- quality open chromatin maps for carefully-staged IVF and SCNT bovine embryos and leveraged cutting-edge single-cell open chromatin profiling to clarify intra- and inter-embryo heterogeneity in chromatin reprogramming. These maps allow us to interrogate, for the first time, how specific regions of the genome open and close during normal preimplantation development, and how SCNT embryos deviate from this paradigm. Our preliminary data clearly indicate early cleavage staged SCNT embryos do not establish open chromatin properly, and implicate an important contributor to EGA, a transcription factor (TF) DUX, as a major deficiency in SCNT embryos. We hypothesize that failures in chromatin reprogramming during SCNT preimplantation development manifest later in postimplantation development, and that targeted intervention using TF expression or preimplantation screening will improve SCNT developmental success. Aim 1 consists of three subaims: 1.1. Determine sex-specific SCNT open chromatin regions that deviate from IVF embryos, or exhibit sex-specificity; 1.2. Identify somatic memory in SCNT embryos that is resistant to reprogramming using DNA methylation profiling; and 1.3. Identify gene expression differences between SCNT and IVF blastocysts and evaluate if they are biased towards ICM or TE lineages. Based on these data, we will use bioinformatic analyses to identify additional TFs/regulators for further mechanistic dissection. In Aim 2, three subaims will be tested: 2.1. if ectopic enforcement of EGA (using DUX and other TFs) can reactivate the EGA program in cultured fibroblasts; 2.2. If targeted activation of the EGA network in SCNT embryos using TF or TF combinations (from 2.1) would improve reprogramming and facilitate their development and 2.3. If single- cell biopsy and open chromatin profiling at early stages can predict developmental outcomes of both IVF and SCNT embryos. These goals will improve the efficiency of generation of agricultural and biomedical animal models and establish strategies and insights for human clinical applications.